The goal of our studies is to develop and test rotavirus DNA vaccines in mice and gnotobiotic pigs that will provide the basis for development of human rotavirus DNA vaccines. Rotaviruses are the major cause of severe acute diarrhea in children, resulting in an estimated 870,000 deaths per year, and current vaccines have not been effective in developing countries where the need is the greatest. We have shown that protective immunity in mice was obtained by parenteral inoculation of DNA vaccines encoding rotavirus proteins and by oral administration of rotavirus DNA vaccines encapsulated in poly (lactide-co-glycolide) (PLG) microparticles. We will evaluate protection generated by DNA vaccines in mice and in gnotobiotic or rotavirus antibody-free conventional pigs, the only animal models that can be clinically infected with human rotaviruses. Immunization will be by oral administration or by intramuscular inoculation with VP2, VP4, VP6 and VP7 DNA vaccines. VP7 is the major neutralization antigen of the outer capsid and the VP7 (G) serotypes 1-4 are the basis of the live, attenuated rhesus-human reassortant tetravalent rotavirus vaccine. To test the possibility of preparing VP7 DNA vaccines derived from human and simian rotaviruses, we will prepare one VP7 DNA vaccine from simian SA-11 rotavirus (for testing against EDIM rotavirus in the mouse model; EDIM and SA-11 are both G serotype 3 viruses), and one VP7 DNA vaccine from human Wa rotavirus (G serotype 1) for testing against Wa rotavirus challenge in gnotobiotic pigs. Mucosal and systemic immune responses to DNA vaccines will be examined in an adult mouse model and in gnotobiotic pigs or rotavirus antibody-free conventional pigs. The antibodies and specific isotypes induced by each DNA vaccine will be examined for virus neutralizing activity and epitope specificity. Cell-mediated immune responses to be examined in the mouse model include cytotoxic T cell responses and specific T helper cell subsets induced. Studies in pigs will include quantitating lymphoproliferative (T cell) and antibody secreting cell (B cell) responses from mucosal and systemic tissues. The need for improved rotavirus vaccines is a continuing one. Immunization with DNA vaccines that express specific rotaviral proteins offers a new approach to vaccination against rotaviruses and may provide the next generation of rotavirus vaccines.